Abstract

Based on observations of the mechanism underlying target discrimination, researchers have developed highly accurate variants of CRISPR-Cas9.

Genome editing has rapidly become a promising and powerful approach for the treatment of inherited disease, largely due to the development of CRISPR-Cas9, a bacterial-derived enzyme capable of creating double strand breaks at specific DNA sequences. However, off-target cutting at DNA sites similar to the targeted sequence is considered a significant hurdle to successful clinical translation of Cas9-based approaches. In their recent article, Chen et al. have made fundamental discoveries about the mechanism by which Cas9 recognizes a target and proceeds through a biological checkpoint to cleave DNA. Then, using this knowledge, they designed a new hyper-accurate variant of Cas9 with improved specificity and retained on-target activity.

The group analyzed previously designed, high-fidelity versions of Cas9 to better understand the mechanism by which they achieved reduced off-target cleavage. First, they discovered that contrary to one possible hypothesis, Cas9 variants are bound to DNA with an affinity similar to the WT version. Using biophysical methods, the authors further illuminated the mechanism by which WT Cas9 achieves DNA target discrimination. The process involves allosteric recognition of target binding by a domain of the enzyme called REC3, leading to changes in the position of a second domain, REC2. This in turn enables a third domain, HNH, to dock in an active conformation and also activates the RuvC nuclease portion of the enzyme, which together cut through double-stranded DNA.

The authors also noted that the HNH domains of high fidelity versions of Cas9 are more sensitive to base-pair differences than WT Cas9, and they remain in a bound but inactive conformation in the presence of mismatches. Mutations in the REC3 region, they determined, lead to the HNH domain being stuck more stringently at the checkpoint phase. Building on this information, they generated a cluster of mutations in the REC3 domain to create HypaCas9, which has increased fidelity. Together, these experiments further illuminate the mechanism by which Cas9 recognizes and cleaves specific DNA sequences, and build toward important advances in increased safety and specificity of CRISPR-Cas9 cleavage that are crucial for clinical translation of genome editing treatments.